U.S. patent application number 16/061524 was filed with the patent office on 2020-08-20 for use of adjuvants for the prevention and/or treatment of autoimmune diseases.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS, SA. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS, SA. Invention is credited to Sandra MOREL, Charlotte Veronique POUCHY, Benoit Laurent SALOMON, Nathalie Raoul Liliane VANERHEYDE.
Application Number | 20200261569 16/061524 |
Document ID | 20200261569 / US20200261569 |
Family ID | 1000004812524 |
Filed Date | 2020-08-20 |
Patent Application | download [pdf] |
United States Patent
Application |
20200261569 |
Kind Code |
A1 |
MOREL; Sandra ; et
al. |
August 20, 2020 |
USE OF ADJUVANTS FOR THE PREVENTION AND/OR TREATMENT OF AUTOIMMUNE
DISEASES
Abstract
The invention provides an adjuvant for use in the prevention
and/or treatment of an autoimmune disease.
Inventors: |
MOREL; Sandra; (Rixensart,
BE) ; POUCHY; Charlotte Veronique; (Paris, FR)
; SALOMON; Benoit Laurent; (Parai, FR) ;
VANERHEYDE; Nathalie Raoul Liliane; (Rixensart, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS, SA |
Paris |
|
FR |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS,
SA
Rixensart
BE
SORBONNE UNIVERSITE
Paris
FR
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE
MEDICALE
Paris
FR
|
Family ID: |
1000004812524 |
Appl. No.: |
16/061524 |
Filed: |
December 15, 2016 |
PCT Filed: |
December 15, 2016 |
PCT NO: |
PCT/EP2016/081181 |
371 Date: |
June 12, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/107 20130101;
A61P 37/02 20180101; A61K 2039/55555 20130101; A61K 2039/55577
20130101; A61K 39/39 20130101; A61K 9/127 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; A61K 9/107 20060101 A61K009/107; A61K 9/127 20060101
A61K009/127; A61P 37/02 20060101 A61P037/02 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2015 |
GB |
1522329.0 |
Claims
1. An adjuvant for use in the prevention and/or treatment of an
autoimmune disease, wherein the adjuvant does not comprise
aluminium and is suitable for use in a human subject.
2. The adjuvant of claim 1, wherein the adjuvant comprises an
immunologically active saponin.
3. The adjuvant of claim 2, wherein the adjuvant comprises
QS21.
4. The adjuvant of claim 2, wherein the adjuvant comprises a
sterol.
5. The adjuvant of claim 1, wherein the adjuvant comprises a TLR
agonist, such as a TLR4 agonist.
6. The adjuvant of claim 1, wherein the adjuvant comprises
3D-MPL.
7. The adjuvant of claim 1, wherein the adjuvant comprises QS21 and
3D-MPL in a liposomal formulation.
8. The adjuvant of claim 1, wherein the adjuvant comprises an
oil-in-water emulsion.
9. The adjuvant of claim 8, wherein the emulsion comprises
squalene.
10. The adjuvant of claim 9, wherein the emulsion further comprises
alpha-tocopherol and polysorbate 80 in an aqueous phase.
11. The adjuvant of claim 1, wherein the autoimmune disease is a
disease selected from the group consisting of: multiple sclerosis,
type 1 diabetes, rheumatoid arthritis, inflammatory bowel diseases,
systemic lupus erythematosus, psoriasis, scleroderma, and
autoimmune thyroid diseases.
12. The adjuvant of claim 1, wherein the autoimmune disease is a
disease affecting the nervous system, such as the central nervous
system.
13. The adjuvant of claim 1, wherein the autoimmune disease is
multiple sclerosis.
14. The adjuvant of claim 1, wherein the autoimmune disease is
acute disseminated encephalomyelitis.
15. The adjuvant of claim 1, wherein the autoimmune disease is type
1 diabetes.
16. The adjuvant of claim 1, wherein the adjuvant is for use in a
human patient.
17. The adjuvant of claim 1, wherein the adjuvant is for use in the
prevention of an autoimmune disease in a human patient who is
pre-disposed to develop an autoimmune disease.
18. A method for preventing and/or treating an autoimmune disease
comprising administration of an adjuvant to a subject, wherein the
adjuvant does not comprise aluminium and is suitable for use in a
human subject.
19. (canceled)
20. Use of an adjuvant in the manufacture of a medicament for the
prevention and/or treatment of an autoimmune disease, wherein the
adjuvant does not comprise aluminium and is suitable for use in a
human subject.
21. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to adjuvants, in particular to
the use of adjuvants in the prevention and/or treatment of
autoimmune diseases.
BACKGROUND OF THE INVENTION
[0002] In many vaccines, antigens are in the form of purified or
subunit proteins to improve their safety, but this comes at the
expense of lower immunogenicity. Therefore, effective adaptive
immune responses require the addition of adjuvants to trigger early
inflammation and activate antigen presenting cells.
[0003] Aluminium adjuvant (alum) is widely used to promote antibody
responses by inducing a Th2 response. Squalene-based adjuvants MF59
and adjuvant system (AS) 03 promote both Th1 and Th2 responses
through their capacity to induce chemokine and cytokine release,
leading to massive recruitment and activation of immune cells. As
for the Toll-like-receptor ligand-based adjuvants AS04 and AS01,
they induce Th1 and Th2 responses by directly activating innate
immune cells.
[0004] Beyond the effects on effector responses, some studies
revealed intriguing and paradoxical observations of the
immunomodulatory impact of certain adjuvants in autoimmune
diseases. Administration of complete Freund's adjuvant (CFA), used
only in veterinary vaccines, induced protection and remission of
type 1 diabetes in non-obese diabetic mice (Sadelain et al. (1990)
Diabetes 39: 583). Also, administration of lipopolysaccharides
(LPS), a natural TLR4 ligand used in research trials for veterinary
vaccines, induced protection from experimental autoimmune
encephalomyelitis (EAE), a mouse model of multiple sclerosis
(Buenafe et al. (2007) J. Neuroimmunol. 182: 32). While these are
interesting observations, both CFA and LPS are not suitable for
human use because of toxicity.
[0005] Autoimmune diseases are caused by an immune response against
constituents of the body's own tissues. More than 80 autoimmune
diseases are known. Examples of autoimmune diseases include e.g.
rheumatoid arthritis (RA), systemic lupus erythematosus (lupus),
inflammatory bowel disease (IBD), multiple sclerosis (MS), type 1
diabetes mellitus, Guillain-Barre syndrome, Crohn's disease and
psoriasis. Many of these diseases are chronic and can cause
significant morbidity and disability. Treatment of autoimmune
diseases is generally based on immunosuppression. While significant
progress has been made in the treatment of autoimmune diseases,
there is still a need for improved products and methods, which
increase efficacy, reduce side-effects, are easy to administer,
safe and can be used in long-term or even chronic treatment of
these diseases.
SUMMARY OF THE INVENTION
[0006] It has now surprisingly been found that AS01 and AS03, two
non-alum containing adjuvants suitable for, and approved for, human
use, can induce almost complete prevention of autoimmune disease in
an animal model, supporting their use in the prevention and/or
treatment of autoimmune diseases in humans.
[0007] Accordingly, in a first aspect, the invention relates to an
adjuvant for use in the prevention and/or treatment of an
autoimmune disease, wherein the adjuvant does not comprise
aluminium and is suitable for use in a human subject.
[0008] In a further aspect, there is provided a method for
preventing and/or treating an autoimmune disease comprising
administration of an adjuvant to a subject, wherein the adjuvant
does not comprise aluminium and is suitable for use in a human
subject.
[0009] In an even further aspect, the invention relates to the use
of an adjuvant in the manufacture of a medicament for the
prevention and/or treatment of an autoimmune disease, wherein the
adjuvant does not comprise aluminium and is suitable for use in a
human subject.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1: a, Representative picture of popliteal draining
lymph nodes (dLN) 4 days after subcutaneous injection (footpads) of
vaccine adjuvants. b, Typical flow cytometry analysis of Treg
cells. c, Fold-increase of the percentage of Foxp3+ cells among
CD4+ cells, compared to mice injected with PBS, in pLN 4 days and 7
days after subcutaneous injection of vaccine adjuvants. Cumulative
data from 3 independent experiments. d, CD4+Foxp3-CD90.1+cells were
adoptively transferred prior to adjuvant injection and induction of
Foxp3 on donor cells was evaluated at day 4 as a marker of pTreg
cells. Represented is an example of analysis of Treg induction
following AS01 treatment. e,f, Ex vivo suppressive activity of Treg
cells purified from dLN of mice treated with AS01 4 days earlier.
Representative data at a 1 to 2 Treg:Tconv cell ratio (e) and mean
.+-.SEM at different Treg:Tconv cell ratio from 4 independent
experiments M. "Treg" cells is for regulatory T cells and "Tconv"
cells is for conventional T cells.
[0011] FIG. 2: a, Absolute number of CD45.sup.+ cells in dLN 4 days
after subcutaneous injection of vaccine adjuvants. Cumulative data
from 3 independent experiments. b, Representative expression of
CD44, ICOS and KI-67 in Treg cells from dLN 4 days after
subcutaneous injection of AS01. c, In vitro suppressive activity of
Treg cells purified from dLN 3 days after subcutaneous injection of
vaccine adjuvants. d, Representative Foxp3 expression in Treg cells
from dLN 3 days after subcutaneous injection of AS01. e, In vitro
suppressive activity of Treg cells purified from dLN 7 days after
subcutaneous injection of AS01. "Treg" cells is for regulatory T
cells.
[0012] FIG. 3: a, Clinical score of EAE (experimental autoimmune
encephalomyelitis) in mice immunized to induce EAE at day 0 and
treated with adjuvants at day -3 and 0. Cumulative data from 3
independent experiments. b, Representative proliferation (from 2
independent experiments) of 2D2 MOG.sub.35-55-specific T cell 3
days after transfer in mice immunized to induce EAE (day 0) and
treated with adjuvants (day -3). c, INF-.gamma. and IL-17
production by MOG-reactive T cells from dLN of mice immunized 10
days earlier to induce EAE (day 0) and treated with AS01 or AS03
(day -3). d, Clinical score of EAE in mice immunized to induce EAE
at day 0 and transferred the day before with Treg cells purified
from PBS, AS01 or AS03 treated mice. e,f Percentage of Treg cells
(e) and of integrin .alpha.L.sup.+ and .alpha.M.sup.+ bamong Treg
and Tconv cells (f) in dLN at day 10 in mice immunized to induce
EAE at day 0 and treated with AS03 at day -3. "Treg" cells is for
regulatory T cells and "Tconv" cells is for conventional T
cells.
[0013] FIG. 4: a, b Clinical score of EAE (experimental autoimmune
encephalomyelitis) in mice immunized to induce EAE at day 0 and
treated with AS01 or AS03 at day -3 (a) or day 0 (b). Cumulative
data from 3 independent experiments. c,d Proportions of
CCR6.sup.+cells and CXCR3.sup.+ cells (c) and of integrin
.alpha.4.sup.+ cells and integrin .alpha.L.sup.+ cells (d) among
Treg cells and Tconv cells in dLN at day 10 in mice immunized to
induce EAE at day 0 and treated with AS03 at day 0 and -3. "Treg"
cells is for regulatory T cells and "Tconv" cells is for
conventional T cells.
DETAILED DESCRIPTION
[0014] As described above, in a first aspect, the invention relates
to an adjuvant for use in the prevention and/or treatment of an
autoimmune disease, wherein the adjuvant does not comprise
aluminium and is suitable for use in a human subject.
[0015] Typically, the aim of the use of the invention, or of the
method of the invention, is to prevent an autoimmune disease,
including delaying onset of the disease, and/or treat such a
disease, i.e. reduce the severity of such a disease, e.g. by
reducing the cause of the autoimmune disease and/or reducing its
symptoms. In one embodiment, a reduction of symptoms of more than
50%, such more than 75%, as determined according to the Examples
herein, is achieved.
Adjuvants for Use in the Invention
[0016] In some embodiments, the adjuvant comprises an
immunologically active saponin, e.g. QS21. Adjuvants comprising
saponins have been described in the art. Saponins are e.g.
described in: Lacaille-Dubois and Wagner (1996) (A review of the
biological and pharmacological activities of saponins.
Phytomedicine Vol. 2: 363). Saponins are known as adjuvants in
vaccines. For example, Quil A (derived from the bark of the South
American tree Quillaja Saponaria Molina), was described by
Dalsgaard et al. (1974) in "Saponin adjuvants" (Archly. fur die
gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, 243) to
have adjuvant activity. Purified fractions of Quil A have been
isolated by HPLC which retain adjuvant activity without the
toxicity associated with Quil A (Kensil et al. (1991) J. Immunol.
146: 431). Quil A fractions are also described in U.S. Pat. No.
5,057,540 and "Saponins as vaccine adjuvants" (Kensil, C. R., Crit.
Rev. Ther. Drug Carrier Syst., 1996, 12 (1-2): 1-55).
[0017] Two such fractions, suitable for use in the present
invention, are QS7 and QS21 (also known as QA-7 and QA-21). QS21 is
a preferred immunologically active saponin fraction for use in the
present invention. QS21 has been reviewed in "Vaccine Adjuvants:
preparation methods and research protocols" (Humana Press, Totowa,
N.J., Edited by Derek T. O'Hagan, Chapter 15: "QS21 Adjuvant").
Particulate adjuvant systems comprising fractions of Quil A, such
as QS21 and QS7, are e.g. described in WO 96/33739, WO 96/11711 and
WO 07/068907.
[0018] In addition to the saponin component, the adjuvant
preferably comprises a sterol. The presence of a sterol may further
reduce reactogenicity of compositions comprising saponins, see e.g.
EP0822831. Suitable sterols include beta-sitosterol, stigmasterol,
ergosterol, ergocalciferol and cholesterol. Cholesterol is
particularly suitable. Suitably, the immunologically active saponin
fraction is QS21 and the ratio of QS21:sterol is from 1:100 to 1:1
w/w, such as from 1:10 to 1:1 w/w, e.g. from 1:5 to 1:1 w/w.
[0019] In some embodiments, the adjuvant used in the invention
comprises a TLR (Toll-like receptor) agonist, such as a TLR4
agonist, e.g. 3D-MPL. The use of TLR agonists in adjuvants is
well-known in art and has been reviewed e.g. by Lahiri et al.
(2008, Vaccine 26: 6777). TLRs that can be stimulated to achieve an
adjuvant effect include TLR2, TLR4, TLR5, TLR7, TLR8 and TLR9.
TLR2, TLR4, TLR7 and TLR8 agonists, particularly TLR4 agonists, are
preferred.
[0020] Suitable TLR4 agonists include lipopolysaccharides, such as
monophosphoryl lipid A (MPL) and 3-O-deacylated monophosphoryl
lipid A (3D-MPL). U.S. Pat. No. 4,436,727 discloses MPL and its
manufacture. U.S. Pat. No. 4,912,094 and reexamination certificate
B1 4,912,094 discloses 3D-MPL and a method for its manufacture.
Another TLR4 agonist is glucopyranosyl lipid adjuvant (GLA), a
synthetic lipid A-like molecule (see e.g. Fox et al. (2012) Clin.
Vaccine Immunol. 19: 1633). In a further embodiment, the TLR4
agonist may be a synthetic TLR4 agonist, such as a synthetic
disaccharide molecule, similar in structure to MPL and 3D-MPL, or
may be synthetic monosaccharide molecules, such as the aminoalkyl
glucosaminide phosphate (AGP) compounds disclosed in, for example,
WO 98/50399, WO 01/34617, WO 02/12258, WO 03/065806, WO 04/062599,
WO 06/016997, WO 06/12425, WO 03/066065, and WO01/90129. Such
molecules have also been described in the scientific and patent
literature as lipid A mimetics. Lipid A mimetics suitably share
some functional and/or structural activity with lipid A, and in one
aspect are recognised by TLR4 receptors. AGPs as described herein
are sometimes referred to as lipid A mimetics in the art. In a
preferred embodiment, the TLR4 agonist is 3D-MPL. TLR4 agonists,
such as 3-O-deacylated monophosphoryl lipid A (3D-MPL), and their
use as adjuvants in vaccines has e.g. been described in WO 96/33739
and WO 07/068907 and reviewed in Alving et al. (2012, Curr. Opin.
in Immunol. 24: 310).
[0021] In a preferred embodiment of the invention, the adjuvant
comprises both an immunologically active saponin and a TLR4
agonist, e.g. QS21 and 3D-MPL.
[0022] In a further preferred embodiment, the adjuvant comprises an
immunologically active saponin and a TLR4 agonist, e.g. QS21 and
3D-MPL, in a liposomal formulation.
[0023] The term "liposome" when used herein refers to uni- or
multilamellar lipid structures enclosing an aqueous interior.
Liposomes and liposome formulations are well known in the art.
Liposomal presentations are e.g. described in WO 96/33739 and WO
07/068907. Lipids which are capable of forming liposomes include
all substances having fatty or fat-like properties.
[0024] Liposome size may vary from 30 nm to several pm depending on
the phospholipid composition and the method used for their
preparation. In particular embodiments of the invention, the
liposome size will be in the range of 50 nm to 500 nm, and in
further embodiments, in the range of 50 nm to 200 nm. Dynamic laser
light scattering is a method used to measure the size of liposomes
well known to those skilled in the art.
[0025] In a particularly suitable embodiment, liposomes used in the
invention comprise DOPC and a sterol, in particular cholesterol.
Thus, in a particular embodiment, adjuvants of the invention
comprise QS21 in any amount described herein in the form of a
liposome, wherein said liposome comprises DOPC and a sterol, in
particular cholesterol.
[0026] In one embodiment, the adjuvant comprises between 5 and 100,
such as between 10 and 75, e.g. 25 or 50 .mu.g, of QS21 per dose
and between 5 and 100, such as between 10 and 75, e.g. 25 or 50
.mu.g of 3D-MPL per dose.
[0027] It is well known that for parenteral administration,
solutions should be physiologically isotonic (i.e. have a
pharmaceutically acceptable osmolality) to avoid cell distortion or
lysis. An "isotonicity agent" is a compound that is physiologically
tolerated and imparts a suitable tonicity to a formulation (e.g.
immunogenic compositions of the invention) to prevent the net flow
of water across cell membranes that are in contact with the
formulation. Aqueous adjuvant compositions are known which contain
100 mM sodium chloride or more, for example adjuvant system A (AS)
in WO 05/112991 and WO 08/142133 or the liposomal adjuvants
disclosed in WO 07/068907.
[0028] In some embodiments, the isotonicity agent used for the
composition is a salt. In other embodiments, however, the
composition comprises a non-ionic isotonicity agent and the
concentration of sodium chloride or the ionic strength in the
composition is less than 100 mM, such as less than 80 mM, e.g. less
than 30 mM, such as less 10 mM or less than 5 mM. In a preferred
embodiment, the non-ionic isotonicity agent is a polyol, such as
sorbitol. The concentration of sorbitol may be e.g. between about
3% and about 15% (w/v), such as between about 4% and about 10%
(w/v). Adjuvants comprising an immunologically active saponin
fraction and a TLR4 agonist wherein the isotonicity agent is salt
or a polyol have been described in WO 10/142685, see e.g. Examples
1 and 2 in WO 10/142685.
[0029] In one embodiment, the adjuvant used in the invention
comprises an oil-in-water emulsion. Suitably, said emulsion
comprises a metabolisable oil in an amount of 0.5% to 20% of the
total volume,
[0030] The meaning of the term metabolisable oil is well known in
the art. Metabolisable can be defined as `being capable of being
transformed by metabolism` (Dorland's Illustrated Medical
Dictionary, W.B. Sanders Company, 25th edition (1974)). The oil may
be any vegetable oil, fish oil, animal oil or synthetic oil, which
is not toxic to the recipient and is capable of being transformed
by metabolism. Nuts, seeds, and grains are common sources of
vegetable oils. Synthetic oils are also part of this invention and
can include commercially available oils such as NEOBEE.RTM. and
others. A particularly suitable metabolisable oil is squalene.
Squalene
(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an
unsaturated oil which is found in large quantities in shark-liver
oil, and in lower quantities in olive oil, wheat germ oil, rice
bran oil, and yeast, and is a particularly suitable oil for use in
this invention. Squalene is a metabolisable oil by virtue of the
fact that it is an intermediate in the biosynthesis of cholesterol
(Merck index, 10th Edition, entry No. 8619).
[0031] Oil-in-water emulsions per se are well known in the art, and
have been suggested to be useful as adjuvant compositions (EP
399843; WO 95/17210). Suitably, the metabolisable oil is present in
an amount of 0.5% to 20% (final concentration) of the total volume
of the immunogenic composition, suitably an amount of 1.0% to 10%
of the total volume, suitably in an amount of 2.0% to 6.0% of the
total volume.
[0032] In a specific embodiment, the metabolisable oil is present
in a final amount of about 0.5%, 1%, 3.5% or 5% of the total volume
of the immunogenic composition. In another specific embodiment, the
metabolisable oil is present in a final amount of 0.5%, 1%, 3.57%
or 5% of the total volume of the immunogenic composition. A
suitable amount of squalene is about 10.7 mg per dose, suitably
from 10.4 to 11.0 mg per dose.
[0033] Suitably the oil-in-water emulsion systems used in the
present invention have a small oil droplet size in the sub-micron
range. Suitably the droplet sizes will be in the range 120 to 750
nm, suitably sizes from 120 to 600 nm in diameter. Typically the
oil-in water emulsion contains oil droplets of which at least 70%
by intensity are less than 500 nm in diameter, in particular at
least 80% by intensity are less than 300 nm in diameter, suitably
at least 90% by intensity are in the range of 120 to 200 nm in
diameter.
[0034] The oil-in-water emulsion according to the invention may
comprise a sterol or a tocopherol, such as alpha-tocopherol.
Sterols are well known in the art, for example cholesterol is well
known and is, for example, disclosed in the Merck Index, 11th
Edition, page 341, as a naturally occurring sterol found in animal
fat. Other suitable sterols include .beta.-sitosterol,
stigmasterol, ergosterol and ergocalciferol. Suitably
alpha-tocopherol or a derivative thereof, such as alpha-tocopherol
succinate is present. Suitably alpha-tocopherol is present in an
amount of between 0.2% and 5.0% (v/v) of the total volume of the
immunogenic composition, suitably at an amount of 2.5% (v/v) in a
0.5 ml dose volume, or 0.5% (v/v) in 0.5 ml dose volume or 1.7-1.9%
(v/v), suitably 1.8% in 0.7 ml dose volume. By way of
clarification, concentrations given in v/v can be converted into
concentration in w/v by applying the following conversion factor: a
5% (v/v) alpha-tocopherol concentration is equivalent to a 4.8%
(w/v) alpha-tocopherol concentration. A suitable amount of
alpha-tocopherol is about 11.9 mg per dose, suitably from 11.6 to
12.2 mg per dose.
[0035] The oil-in-water emulsion may comprise an emulsifying agent.
The emulsifying agent may be present at an amount of 0.01 to 5.0%
by weight of the immunogenic composition (w/w), suitably present at
an amount of 0.1 to 2.0% by weight (w/w). Suitable concentration
are 0.5 to 1.5% by weight (w/w) of the total composition. The
emulsifying agent may suitably be polyoxyethylene sorbitan
monooleate (polysorbate 80 or Tween 80). In a specific embodiment,
a 0.5 ml dose volume contains 1% (w/w) Tween 80, and a 0.7 ml dose
volume contains 0.7% (w/w) Tween 80. In another specific embodiment
the concentration of Tween 80 is 0.2% (w/w). A suitable amount of
polysorbate 80 is about 4.9 mg per dose, suitably from 4.6 to 5.2
mg per dose.
[0036] Span 85 (polyoxyethylene sorbitan trioleate) may also be
present, for example at a level of 1%. An example of oil-in-water
emulsion adjuvant comprises Span85 for use in the invention is
given and detailed in EP0399843B, also known as MF59.
[0037] In a preferred embodiment, the adjuvant is an oil-in-water
emulsion comprising squalene, alpha-tocopherol and a surfactant,
i.e. the emulsion comprises squalene, alpha-tocopherol and a
surfactant, e.g. polysorbate 80, in an aqueous phase. Preparation
of such adjuvants is e.g. described in WO 95/17210 and WO
06/100109. In one embodiment, the emulsion comprises 2-10% (v/v)
squalene, from 2-10% (v/v) alpha-tocopherol and from 0.3-3% (v/v)
Tween 80. Preferably, the emulsion comprises 2.5% squalene (v/v),
2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan
monooleate (v/v) (Tween 80).
Autoimmune Diseases to be Prevented and/or Treated
[0038] As explained above, the invention relates to the use of
adjuvants for the prevention and/or treatment of autoimmune
diseases. The following diseases have been classified as autoimmune
diseases: Acute disseminated encephalomyelitis, Addison's disease,
Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing
spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome
(APS), Autoimmune hepatitis, Autoimmune inner ear disease (AIED),
Axonal & neuronal neuropathy (AMAN), Behcet's disease, Bullous
pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease,
Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic
recurrent multifocal osteomyelitis (CRMO), Churg-Strauss,
Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan's syndrome,
Cold agglutinin disease, Congenital heart block, Coxsackie
myocarditis, CREST syndrome, Crohn's disease, Dermatitis
herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis
optica), Discoid lupus, Dressler's syndrome, Endometriosis,
Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema
nodosum, Essential mixed cryoglobulinemia, Evans syndrome,
Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal
arteritis), Giant cell myocarditis, Glomerulonephritis,
Goodpasture's syndrome, Granulomatosis with Polyangiitis, Graves'
disease, Guillain-Barre syndrome, Hashimoto's thyroiditis,
Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes
gestationis or pemphigoid gestationis (PG), Hypogammalglobulinemia,
IgA Nephropathy, IgG4-related sclerosing disease, Inclusion body
myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis,
Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome,
Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus,
Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lyme
disease chronic, Meniere's disease, Microscopic polyangiitis (MPA),
Mixed connective tissue disease (MCTD), Mooren's ulcer,
Mucha-Habermann disease, Multiple sclerosis (MS), Myasthenia
gravis, Myositis, Narcolepsy, Neuromyelitis optica, Neutropenia,
Ocular cicatricial pemphigoid, Optic neuritis, Palindromic
rheumatism (PR), PANDAS (Pediatric Autoimmune Neuropsychiatric
Disorders Associated with Streptococcus), Paraneoplastic cerebellar
degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH),
Parry Romberg syndrome, Pars planitis (peripheral uveitis),
Parsonnage-Turner syndrome, Pemphigus, Peripheral neuropathy,
Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, skin changes), Polyarteritis nodosa, Polymyalgia
rheumatic, Polymyositis, Postmyocardial infarction syndrome,
Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary
sclerosing cholangitis, Progesterone dermatitis, Psoriasis,
Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma
gangrenosum, Raynaud's phenomenon, Reactive Arthritis, Reflex
sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis,
Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic
fever, Rheumatoid arthritis (RA), Sarcoidosis, Schmidt syndrome,
Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular
autoimmunity, Stiff person syndrome (SPS), Subacute bacterial
endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),
Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,
Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS),
Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC),
Undifferentiated connective tissue disease (UCTD), Uveitis,
Vasculitis, Vitiligo, and Wegener's granulomatosis (now termed
Granulomatosis with Polyangiitis (GPA).
Preferred Disease to be Prevented and/or Treated According to the
Invention Include: [0039] Rheumatoid Arthritis [0040] In people
with rheumatoid arthritis, the immune system predominantly targets
the lining (synovium) that covers various joints. Inflammation of
the synovium is usually symmetrical (occurring equally on both
sides of the body) and causes pain, swelling, and stiffness of the
joints. These features distinguish rheumatoid arthritis from
osteoarthritis, which is a more common and degenerative
"wear-and-tear" arthritis. Currently available therapy focuses on
reducing inflammation of the joints with anti-inflammatory or
immunosuppresssive medications. Sometimes, the immune system may
also target the lung, blood vessels, or eye; occasionally patients
may also develop symptoms of other autoimmune diseases such as
Sjogren's the inflammation, itching, and scaling. [0041] Multiple
Sclerosis [0042] Multiple sclerosis is a disease in which the
immune system targets nerve tissues of the central nervous system.
Most commonly, damage to the central nervous system occurs
intermittently, allowing a person to lead a fairly normal life. At
the other extreme, the symptoms may become constant, resulting in a
progressive disease with possible blindness, paralysis, and
premature death. Some medications such as beta interferon are
helpful to people with the intermittent form of multiple sclerosis.
In young adults, multiple sclerosis is the most common disabling
disease of the nervous system. [0043] Immune-Mediated or Type 1
Diabetes Mellitus [0044] Type 1 diabetes mellitus results from
autoimmune destruction of the insulin-producing cells of the
pancreas. Insulin is required by the body to keep the blood sugar
(glucose) level under control. High levels of glucose are
responsible for the symptoms and the complications of the disease.
However, most of the insulin-producing cells are destroyed before
the patient develops symptoms of diabetes. Symptoms include
fatigue, frequent urination, increased thirst, and possible sudden
confusion. Type 1 diabetes mellitus is usually diagnosed before the
age of 30 and may be diagnosed as early as the first month of life.
Together with Type 2 diabetes (not considered an autoimmune
disease), diabetes mellitus is the leading cause of kidney damage,
loss of eyesight, and leg amputation. Close control of sugar levels
decreases the rate at which these events occur. [0045] Inflammatory
Bowel Diseases [0046] This medical term is used for both Crohn's
disease and ulcerative colitis, two diseases in which the immune
system attacks the gut (intestine). Patients may have diarrhea,
nausea, vomiting, abdominal cramps, and pain that can be difficult
to control. Illness in afflicted individuals can result from
intestinal inflammation and from side effects of the drugs used for
the disease. For example, daily use of high-dose corticosteroid
(prednisone) therapy, which is needed to control severe symptoms of
Crohn's disease, can predispose patients to infections, bone
thinning (osteoporosis), and fractures. [0047] Systemic Lupus
Erythematosus [0048] Patients with systemic lupus erythematosus
most commonly experience profound fatigue, rashes, and joint pains.
In severe cases, the immune system may attack and damage several
organs such as the kidney, brain, or lung. For many individuals,
symptoms and damage from the disease can be controlled with
available anti-inflammatory medications. [0049] Psoriasis [0050]
Psoriasis is an immune system disorder that affects the skin, and
occasionally the eyes, nails, and joints. Psoriasis may affect very
small areas of skin or cover the entire body with a buildup of red
scales called plaques. The plaques are of different sizes, shapes,
and severity and may be painful as well as unattractive. Bacterial
infections and pressure or trauma to the skin can aggravate
psoriasis. Most treatments focus on topical skin care to relieve
the inflammation, itching, and scaling. [0051] Scleroderma [0052]
This autoimmune disease results in thickening of the skin and blood
vessels. Almost every patient with scleroderma has Raynaud's, which
is a spasm of the blood vessels of the fingers and toes. Symptoms
of Raynaud's include increased sensitivity of the fingers and toes
to the cold, changes in skin color, pain, and occasionally ulcers
of the fingertips or toes. In people with scleroderma, thickening
of skin and blood vessels can result in loss of movement and
shortness of breath or, more rarely, in kidney, heart, or lung
failure. [0053] Autoimmune Thyroid Diseases [0054] Hashimoto's
thyroiditis and Grave's disease result from immune system
destruction or stimulation of thyroid tissue. Symptoms of low
(hypo-) or overactive (hyper-) thyroid function are nonspecific and
can develop slowly or suddenly; these include fatigue, nervousness,
cold or heat intolerance, weakness, changes in hair texture or
amount, and weight gain or loss. The diagnosis of thyroid disease
is readily made with appropriate laboratory tests. The symptoms of
hypothyroidism are controlled with replacement thyroid hormone
pills; however, complications from over- or under-replacement of
the hormone can occur. Treatment of hyperthyroidism requires
long-term anti-thyroid drug therapy or destruction of the thyroid
gland with radioactive iodine or surgery. Both of these treatment
approaches carry certain risks and long-term side effects.
[0055] In a preferred embodiment, the autoimmune disease to be
prevented and/or treated is a disease affecting the nervous system,
such as the central nervous system. In an even more preferred
embodiment, the autoimmune disease is multiple sclerosis. In a
further more preferred embodiment, the autoimmune disease it type 1
diabetes. In a further preferred embodiment, the autoimmune disease
is acute disseminated encephalomyelitis.
[0056] In particular, adjuvants comprising an oil-in-water
emulsion, e.g. comprising squalene, alpha-tocopherol and
polysorbate 80 in an aqueous phase, are preferred for the
prevention and/or treatment of diseases affecting the nervous
system, such as the central nervous system, e.g. multiple
sclerosis.
Treatment options In preferred embodiments, the method of the
invention, or use of the invention, comprises multiple
administrations of the adjuvant, for example, at least 2, at least
3, at least 4, at least 5, or at least 10 administrations of the
adjuvant. In one embodiment, the time interval between each of the
administrations is between 1 day and 6 months, e.g. the interval
may be from 1 week to 1 month between each administration.
[0057] In a preferred embodiment, the subject is a human subject.
The human subject to be treated using the method of the invention
may be of any age. In one embodiment, however, the human subject is
more than 18 years of age when the treatment is initiated. In
further embodiments, the subject is more than 40 years of age, such
as more than 50 years of age, e.g. more than 60 years of age. The
subject may be male or female.
[0058] In one embodiment, the adjuvant is for use in the prevention
of an autoimmune disease in a human subject who is pre-disposed to
develop an autoimmune disease, for example, a subject who is
pre-disposed to develop multiple sclerosis or a subject who is
pre-disposed to develop type 1 diabetes. A human subject who is
pre-disposed to develop an autoimmune disease can, for example, be
a subject who shows early clinical signs of an autoimmune disease,
a subject who has a genetic risk of developing an autoimmune
disease or a subject who has specific auto-antibodies.
[0059] A person pre-disposed to develop multiple sclerosis is e.g.
a person having CIS (clinically isolated syndrome)--a first and
single neurologic episode of inflammation or demyelination in the
central nervous system lasting at least 24 hours, in particular
when lesions consistent with multiple sclerosis are seen on
MRI.
[0060] A person pre-disposed to develop type 1 diabetes is e.g. a
person having auto-antibodies, such as autoantibodies GADA, IA-2A
and/or mIAA (Sosenko et al. (2013) Diabetes Care 36: 2615).
[0061] The adjuvant may be administered via various suitable
routes, including parenteral, such as intramuscular, intradermal or
subcutaneous administration. Suitably, the adjuvant compositions
used in the present invention have a human dose volume of between
0.05 ml and 1 ml, such as between 0.1 and 0.5 ml, in particular a
dose volume of about 0.5 ml, or 0.7 ml.
[0062] The adjuvant may be given as monotherapy or in combination
with other substances, e.g. other substances known to have a
therapeutic or prophylactic effect on autoimmune diseases.
[0063] The teachings of all references in the present application,
including patent applications and granted patents, are herein fully
incorporated by reference. The terms `comprising`, `comprise` and
`comprises` herein are optionally substitutable with the terms
`consisting of`, `consist of`, and `consists of`, respectively. The
invention will be further described by reference to the following,
non-limiting, examples:
EXAMPLES
Example 1--Methods
Mice
[0064] WT mice were purchased from Janvier Labs (France). 2D2
T-cell receptor transgenic mice, specific for myelin
oligodendrocyte glycoprotein, were purchased from the Jackson
Laboratory. Foxp3-IRES-GFP knock-in (Foxp3GFP) mice were kindly
provided by Pr. Bernard Malissen. 2D2 and Foxp3GFP mice were
backcrossed with CD90.1 congenic animals. All mice were on a
C57Bl/6 background. Mice were housed under specific pathogen-free
conditions and were studied at 7-14 week of age. All experimental
protocols were approved by the local ethics committee and are in
compliance with European Union guidelines.
Reagents
[0065] AS01 is composed of MPL (1 mg/ml), QS-21 (1 mg/ml) and
liposomes. AS03 is composed of alpha-tocopherol (23.72 mg/ml),
squalene oil (21.38 mg/ml) and polysorbate 80 (9.72 mg/ml). AS04 is
composed of MPL (0.1 mg/ml) and alum (1 mg/ml). Freund's adjuvants
were purchased from Sigma-Aldrich. Incomplete form (IFA) contains
85% of paraffin oil and 15% of mannide monooleate. Complete form
(CFA) adds Mycobacterium tuberculosis (H37Ra) heat killed and dried
at a concentration of 1 mg/ml.
Administration of Adjuvants
[0066] Mice received 30 .mu.l of vaccine adjuvant or PBS by
subcutaneous route in left hind footpads. To analyze their effect,
left popliteal draining lymph nodes (dLN) and non-draining right
brachial lymph nodes were collected at different time points. In
mice that were immunized to induce EAE (experimental auto-immune
encephalomyelitis), they received 100 .mu.l of vaccine adjuvant or
PBS by subcutaneous route at the base of the tail and at the upper
back 3 days before and on the day of EAE (experimental autoimmune
encephalomyelitis) induction.
Flow Cytometry Analyses
[0067] Cells from lymph nodes were mechanically dissociated and
resuspended in PBS 3% SVF. They were first treated with 2.4G2
antibody to block the Fc receptor and stained with the following
antibodies: anti-CD45 (30-F11), anti-CD3 (145-2C11), anti-CD4
(RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61), anti-Foxp3 (MF23),
anti-CTLA-4 (UC10-4F10-11), anti-GITR (DTA-1), anti-ICOS (17G9),
anti-Ki-67 (B56). All antibodies were obtained from BD Biosciences.
Intracellular staining was performed using the Intracellular
Fixation & Permeabilization Buffer Set from eBioscience. Cells
were acquired on a BD LSRII cytometer and analyzed using Flowio
software.
Treg Peripheral Induction Assessment
[0068] Lymph nodes (brachial, axillary, cervical, and inguinal) and
spleen cells of Foxp3.sup.GFP CD90.1 mice were isolated, treated as
above and stained with an anti-CD4 antibody. CD4.sup.+GFP.sup.-
cells (Tconv) were purified using a BD FACSAria II then
intravenously injected in WT mice (10.sup.6 cells/mouse). The
following day, mice received 30 .mu.l of vaccine adjuvant or PBS by
subcutaneous route in left hind footpads. Three days after, dLN
were collected and cells were stained with anti-CD4, anti-CD90.1
(OX-7), anti-CD90.2 (30-H12) and anti-Foxp3 antibodies. Foxp3
induction in CD90.1 transferred cells was evaluated by flow
cytometry.
In Vitro Treg Suppression Assay
[0069] Foxp3.sup.GFP mice received 100 .mu.l of vaccine adjuvant or
PBS by subcutaneous route at the base of the tail and at the upper
back. Brachial and inguinal lymph nodes were collected at 3 or 7
days. Cells were isolated and stained as described above.
CD4.sup.+GFP.sup.- cells (Tconv) and CD4.sup.+GFP.sup.+ cells
(Treg) were then sorted using BD FACSAria II. Tconv cells were
labeled with CellTrace Violet Proliferation Kit (Life technologies)
and plated in 96-well plate at 2.5.times.10.sup.4 cells/well with
splenocytes from CD3.sup.-/- mice at 7.5.times.10.sup.4 cells/well.
Culture medium was supplemented with anti-CD3 (KT3) from BioXCell
at 0.05 .mu.g/ml. Treg cells were then added at different ratios
from 1:1 to 1:16. At day 3, CellTrace dilution was assessed by flow
cytometry.
EAE Induction
[0070] Mice were immunized by subcutaneous injection of 100 .mu.g
of MOG.sub.35-55 peptide (PolyPeptide) emulsified in 100 .mu.l of
CFA (Sigma-Aldrich) supplemented with 50 .mu.g of heat-killed
Mycobacterium tuberculosis H37Ra (BD Biosciences). Animals were
additionally injected intravenously with 200 ng of Bordetella
pertussis toxin (Enzo) at the time of, and two days following
immunization. The clinical evaluation was performed on a daily
basis by a 5-point scale ranging from 0, no clinical sign; 1, limp
tail; 2, limp tail, impaired righting reflex, and paresis of one
limb; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5,
moribund.
Cytokine Measurement
[0071] Ten days after EAE induction, brachial and inguinal dLN were
collected. Cells were cultured with 1 .mu.g/ml of MOG.sub.35-55
peptide in 96-well plate at 2.times.10.sup.5 cells/well. At day 3,
supernatants were harvested to measure INF-.gamma. and IL-17
secretions by ELISA (eBioscience).
T Cell Priming Evaluation
[0072] Lymph nodes and spleen cells from 2D2 CD90.1 mice were
stained with the following biotin-labeled antibodies: anti-CD19
(6D5), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD8 (53-6.7) and
anti-CD25 (7D4) and then were coated with anti-biotin microbeads
(Miltenyi Biotec). After magnetic sorting, cells of the CD4.sup.+
enriched negative fraction were labeled with CellTrace Violet
Proliferation Kit and were intravenously injected in naive mice
(10.sup.6 cells/mouse). The following day, mice were immunized with
MOG.sub.35-55 peptide as for EAE induction. CellTrace dilution of
CD4.sup.+CD90.1.sup.+V.beta.11.sup.+ (T-cell receptor transgene)
cells was assessed by flow cytometry from brachial and inguinal
lymph nodes at day 3. In mice receiving adjuvants, they were
injected with 100 .mu.l of vaccine adjuvant or PBS by subcutaneous
route at the base of the tail and at the upper back.
Treg Cell Adoptive Transfer
[0073] Foxp3.sup.GFP CD90.1 mice received 100 .mu.l of vaccine
adjuvant or PBS by subcutaneous route at the base of the tail and
at the upper back. At day 3, brachial and inguinal lymph nodes were
collected and cells were stained with anti-CD4 antibody.
CD4.sup.+GFP.sup.+ cells (Treg) were then purified using BD
FACSAria II and intravenously injected in WT mice (1.times.10.sup.6
cells/mouse). The following day, mice were immunized with
MOG.sub.35-55 peptide as previously described in EAE induction.
Statistical Analysis
[0074] Statistical analyses were performed using GraphPad Prism
Software. Statistical significance was determined using the
two-tailed nonparametric Mann-Whitney U test. *p<0.05,
**p<0.01, ***p<0.001. Means.+-.SEM were used throughout the
figures.
Example 2--Vaccine Adjuvants Induce Transient Preferential
Expansion of Treg Cells
[0075] CFA, incomplete Freund's adjuvants (IFA), AS01, AS03 and
AS04 were administered subcutaneously to mice. After 4 days, a
massive inflammation and swelling of the draining lymph nodes (dLN)
was observed (FIG. 1a). The cellularity of these dLN was increased
by 11 to 20 fold (FIG. 2a). By contrast, injection of alum had no
effect on size and cell numbers of dLN.
[0076] Treg cells were then analyzed by flow cytometry (FIG. 1b).
Except for alum, we observed a rapid increase (day 4) of Treg cell
proportions among CD4.sup.+ cells after injection of the different
adjuvants, up to 1.9 fold compared to the PBS-injected mice (FIG.
1c). This Treg cell expansion was associated with an activated
phenotype, as shown by an increased expression of CD44, ICOS and
Ki-67 (FIG. 2b). These Treg cell expansion and activation were
transient since it was no longer present at day 7, except for IFA
(FIG. 1c). Treg cell expansion driven by adjuvant injections was
due to thymic Tregs (tTregs) accumulation and not to increased
induction of peripheral Treg. Indeed, Foxp3 expression was not
induced by adjuvants in adoptively transferred CD4+Foxp3- cells
(FIG. 1d). These data revealed the in vivo activation of tTreg
cells by new generation adjuvants.
[0077] We then assessed the effect of vaccine adjuvant
administration on Treg cell activity by classical in vitro
suppression assay. Alum, Freund's adjuvant, AS03 and AS04 had no
effect on Treg cell function (FIG. 2c), while AS01 partially
reduced their suppressive activity and Foxp3 expression (FIG. 1e
and 1f and 2d). This effect was observed at day 4 but not anymore
at day 7 (FIG. 2e). Thus, administration of new generation
adjuvants did not impair, or only marginally, the suppressive
function of Treg cells.
Example 3--EAE Prevention by Administration of AS01 and AS03
Adjuvants
[0078] In order to evaluate the effects of vaccine adjuvants on
tolerance induction in a physiopathological context, we tested
adjuvant treatments in the EAE autoimmune model. Strikingly, 2
injections of AS01 or AS03, 3 days before and at the time of
immunization, induced almost complete prevention from disease
development. Mean clinical scores were below 0.14-0.17, compared to
1.17 in controls. In contrast, AS04 and alum had no effect on EAE
(FIG. 3a). After only one injection of AS01 or AS03 performed 3
days before, or at the time of immunization, EAE was significantly
delayed or reduced, respectively (FIGS. 4a and 4b). Thus, AS01 and
AS03 have strong preventing effect on EAE.
[0079] We next investigated priming and cytokine polarization of
auto-reactive Tconv cells. T-cell proliferation was assessed after
adoptive transfer of 2D2 transgenic mice expressing a transgenic
I-A.sup.b-restricted T cell receptor specific for MOG.sub.35-55
peptide (Bettelli et al. (2003) J. Exp. Med. 197: 1073) prior to
EAE induction. AS01 administration slightly reduced the
proliferation of MOG.sub.35-55-specific T cells at day 3 in dLN,
whereas AS03 had no effect (FIG. 3b). T-cell polarization was
assessed by measuring the release of IFN.gamma. and IL-17, 2 major
pathogenic cytokines in EAE, at day 10 in dLN after EAE induction
by auto-reactive Tconv cells specific to the MOG.sub.35-55
immunizing peptide. AS01 administration was associated with a
decrease of IFN.gamma. production, whereas IFN.gamma. and IL-17
were unaffected by AS03 administration (FIG. 3c). Thus, although
both AS01 and AS03 administration prevented EAE development, only
AS01 disrupted priming and polarization of MOG.sub.35-55 reactive
T-cells.
[0080] We then performed adoptive transfer experiments to further
analyze the role of Treg cells in adjuvant-driven EAE prevention.
Purified Treg cells from dLN of adjuvant treated mice were injected
into naive mice prior to EAE induction. Amazingly, mice that
received Treg cells from AS03 treated mice were fully protected
from the disease. In contrast, Treg cells from AS01 treated mice
had no effect since recipients mice transferred with these cells
developed the same EAE as control mice transferred with PBS or Treg
from naive mice (FIG. 3d). These data reveal that AS03
administration strongly enhanced the capacity of Treg cells to
control EAE.
[0081] To gain more insight into the mechanism of EAE prevention by
AS03, we further studied Treg cells and molecules involved in
T-cell migration. Cells from dLN of mice immunized to induce EAE
and treated with AS03 were analyzed at day 10. Treg cell proportion
was significantly increased in AS03 treated mice compare to control
(FIG. 3e). The expression of the CCR6 and CXCR3 chemokine receptors
by Tconv cells, which play a critical role in the entry of
pathogenic T cells into the central nervous system (CNS) during EAE
(Reboldi et al. (2009) Nat. Immunol. 10: 514; Sporici and Issekutz
(2010) Eur. J. Immunol. 40: 2751), was unaffected by the AS03
treatment (FIG. 4c). Interestingly, the expression levels of
integrins involved in CNS homing (Yednock et al. (1992) Nature 356:
63; Rothhammer et al. (2011) J. Exp. Med. 208: 2465) were
significantly modified by the adjuvant. Treg cells expressed a
higher level of integrin .alpha.L while Tconv cells showed a
decreased level of integrin .alpha.M (FIG. 3f). Moreover,
expression levels of integrins .alpha.4 and .alpha.L by Tconv cells
were unaffected (FIG. 4d). These results suggest that AS03
administration impacts on migration of Treg and Tconv cells in the
CNS, which may explain its capacity to suppress EAE.
CONCLUSION
[0082] This work demonstrates that some of the new generation
vaccine adjuvants have strong immuno-regulatory properties and
impact on Treg cell activation and function, thus revealing their
potential in the treatment of autoimmune disorders. Both AS01 and
AS03 administration induced protection from EAE. Interestingly,
their suppressive mechanism appears different. AS01 altered priming
and cytokine polarization of encephalitogenic Tconv cells whereas
AS03 strongly increased the protective capacity of Treg cells in
EAE and may modify the migration of Tconv and Treg cells.
* * * * *